Reversible dimerization of a CLC transporter: A model for membrane protein foldin

CLC 转运蛋白的可逆二聚化：膜蛋白折叠模型

• 批准号: 8278841
• 负责人: Janice L Robertson
• 金额: $ 9万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-06-01 至 2014-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8278841
• 关键词: Affinity; Alanine; Amino Acids; Archaea; Back; Biochemical; Biocompatible Materials; Biological Assay; Biological Models; Burial; CLC Gene; Cell physiology; Chemicals; Crystallography; Databases; Dependency; Detergents; Dimerization; Energy Transfer; Entropy; Environment; Ethers; Excision; Fluorescence; Foundations; Free Energy; Freedom; Head; Integral Membrane Protein; Ion Transport; Lead; Length; Lipid Bilayers; Lipid Binding; Lipids; Liposomes; Maps; Measurement; Measures; Membrane; Membrane Lipids; Membrane Proteins; Methods; Micelles; Modeling; Molecular; Mutation; Nature; Phospholipids; Physiological Processes; Population; Positioning Attribute; Process; Property; Proteins; Quality Control; Scanning; Side; Signal Transduction; Solvents; Specificity; Structure; Study models; Surface; System; Temperature; Testing; Thermodynamics; Translating; Tryptophan; Water; Work; alpha helix; antiporter; aqueous; design; dimer; driving force; enthalpy; experience; insight; monomer; protein folding; protein function; protein protein interaction; research study; scaffold; single molecule; therapeutic target; van der Waals force

DESCRIPTION (provided by applicant): The central enigma of protein folding lies in how the physical forces of nature drive a simple string of amino acids into a stable, conformationally defined protein. For soluble proteins, the burial of hydrophobic groups away from aqueous interfaces is a major driving force, but membrane-embedded proteins cannot experience hydrophobic forces, as the lipid bilayer lacks water. A fundamental conundrum thus arises: how does a greasy protein surface find its greasy protein partner in the greasy lipid bilayer to fold faithfully into its native structure? Recently, a structurally stable and functional monomeric form of the normally homodimeric Cl-/H+ antiporter CLC-ec1 was designed by introducing tryptophan mutations at the dimer interface. Preliminary studies show that the protein can be shifted back to the dimer state with additional mutations or in certain lipid conditions. These results present CLC-ec1 as a model for the study of reversible dimerization, which simplifies the protein folding process while still encompassing all of the thermodynamic properties of protein interactions in the membrane environment. To make these energetic measurements, the monomer/dimer populations will be quantified using three well-established methods: (i) ¿Poisson-counting¿ of monomer vs. dimers in liposome populations, (ii) fluorescence self-quenching in liposomes, and (iii) Forster resonance energy transfer (FRET) in liposomes and supported bilayers for single molecule studies. With these assays in place, experiments will be carried out to investigate two alternative hypotheses that have pervaded discourse in this field. First, that specific transmembrane helix interactions are enthalpy-driven by van der Waals forces at highly complementary surfaces. Changes in free energy will be measured upon substitution of interface residues to alanine or tryptophan, with significant positions studied further by increasing side- chain volume to modulate the van der Waals interactions. The second hypothesis is that interactions are driven by increased entropy of lipids upon helix association. To study this, the molecules forming the lipid solvent will be modified by changing the chemical head group, chain length and chain order using unsaturated or tetra-ether lipids from archaea. For all experiments, free energy relationships will also be measured with respect to temperature to extrapolate values for enthalpy and entropy. These results will provide insight into the driving forces for membrane protein interactions, and may even provide a foundation for attacking general questions underlying protein folding in the strange solvent that is the lipid bilayer. PUBLIC HEALTH RELEVANCE: Membrane proteins are molecular "gate-keepers" regulating the passage of biological materials across the lipid bilayer. As such, they are critically involved in physiological processes and may be key therapeutic targets. By understanding the energetic factors governing how these proteins interact and assemble in the lipid environment, we will gain insight into methods of modulating membrane protein function and cell physiology.

描述（由申请人提供）：蛋白质折叠的核心谜团在于自然的物理力如何驱动一串简单的氨基酸变成稳定的、构象确定的蛋白质。对于可溶性蛋白质，远离水界面的疏水基团的埋藏是主要驱动力，但膜嵌入的蛋白质不能经历疏水力，因为脂质双层缺乏水。因此出现了一个基本难题：油脂蛋白表面如何在油脂双层中找到其油脂蛋白伴侣，以忠实地折叠成其天然结构？最近，一种结构稳定且功能齐全的单体形式 通常同型二聚体 Cl-/H+ 逆向转运蛋白 CLC-ec1 的设计是通过在二聚体界面引入色氨酸突变来设计的。初步研究表明，通过额外的突变或在某些脂质条件下，该蛋白质可以转变回二聚体状态。这些结果将 CLC-ec1 作为研究可逆二聚化的模型，它简化了蛋白质折叠过程，同时仍然涵盖了膜环境中蛋白质相互作用的所有热力学特性。为了进行这些能量测量，将使用三种成熟的方法对单体/二聚体群体进行量化：（i）脂质体群体中单体与二聚体的“泊松计数”，（ii）脂质体中的荧光自猝灭，以及（iii）脂质体中的福斯特共振能量转移（FRET）和用于单分子研究的支持双层。完成这些分析后，将进行实验来研究该领域广泛讨论的两种替代假设。首先，特定的跨膜螺旋相互作用是由高度互补的表面上的范德华力驱动的。将界面残基替换为丙氨酸或色氨酸后测量自由能的变化，通过增加侧链体积以调节范德华相互作用来进一步研究重要位置。第二个假设是相互作用是由螺旋缔合时脂质熵增加驱动的。为了研究这一点，将使用来自古细菌的不饱和或四醚脂质通过改变化学头基、链长和链序来修饰形成脂质溶剂的分子。对于所有实验，还将测量相对于温度的自由能关系，以推断出焓和熵的值。这些结果将提供对驾驶的深入了解 膜蛋白相互作用的力，甚至可能为解决脂双层这一奇怪溶剂中蛋白质折叠的一般问题提供基础。 公共健康相关性：膜蛋白是调节生物材料穿过脂质双层的分子“看门人”。因此，他们至关重要 生理过程中，可能是关键的治疗靶点。通过了解控制这些蛋白质如何在脂质环境中相互作用和组装的能量因素，我们将深入了解调节膜蛋白功能和细胞生理学的方法。